Salamanders are important vertebrate model organisms in biomedical research. The Salamander Genome Project (SGP) is developing, applying, and making available genomic and bioinformatics resources for salamanders and in particular the Mexican axoloti (Ambystoma mexicanum). This renewal application proposes two objectives to further enhance axoloti genome resources. First, a 3x Bacterial Artificial Chromosome (BAC) library will be created and screened to identify clones that contain axoloti genes of interest. This list of clones will be made available via the SGP website (Sal-Site) and 25 BACs will be prioritized by the community for isolation and sequencing. A second objective will innovate an approach to sequence the large axoloti genome. Whole axoloti chromosomes that correspond to the third largest chromosome (Chr3) in the karyotype will be isolated by laser capture microscopy. DNA fragment libraries will be generated from the captured chromosomes and these will be sequenced to yield a Chr3 draft assembly, whose contiguity and conceptual context will be increased by integrating paired-end sequences from fosmids, protein-coding sequences deriving from expressed sequence tags, and markers defining linkage group 3 of the Ambystoma meiotic map. Accomplishment of these objectives will yield resources to enable functional studies of genes and epigenetic mechanisms. Also, the project will innovate a 'one-chromosome-at- a time' approach to sequence the large axoloti genome. As a final objective of the application, a historically significant collection of axolotis in the Ambystoma Genetic Stock Center (AGSC) will be maintained and distributed to NIH-funded researchers. Since 1969, the AGSC axoloti collection has been supported by funds received from the National Science Foundation. An increasing number of NIH researchers are requesting axoloti living stocks for regeneration research and at the same time, there is need to develop a stable, long term funding model to maintain this irreplaceable resource. A dual NIH-NSF funding model is proposed where costs associated with the maintenance and distribution of axoloti stocks will be split equally between the NSF and NIH.